Quarter Milk Research Articles

Diversity and Resistance Profiles of ESBL-Producing Gram-Negative Bacteria from Dairy Farms in Southern Türkiye

Background/Objectives: The increasing occurrence of extended-spectrum β-lactamase (ESBL)–producing Enterobacterales, most commonly Escherichia coli, has become a serious problem. The aim of this study was to determine the presence of ESBL-producing Gram-negative bacteria in dairy cattle, goat and sheep farms located in southern Türkiye. Methods: Samples (409 quarter milk samples and 110 fresh faecal samples from cattle, 75 bulk tank milk samples and 225 rectal swab samples from goats and sheep) were subjected to selective isolation on MacConkey agar with ceftazidime (2 µg/mL). Isolates were identified by MALDI-ToF MS. The antimicrobial susceptibility profile of the isolates was determined by the broth microdilution method. To obtain a deeper insight into the genetic diversity of isolates substantially contributing to an efficient spread of their ESBL-determinants (23-MO00001: an E. coli from mastitis and 23-MO00002 Citrobacter freundii), the transmission potential and the genetic background of the plasmid carrying the blaCTX-M determinant was studied with whole genome analysis using Illumina sequencing. Results: Of the samples tested, 47 from the bovine faecal samples, 1 from the subclinical mastitis milk sample, 9 from the goat/sheep rectal swab samples and 5 from the goat/sheep bulk tank milk samples had ceftazidime-resistant Gram-negative strains with the ESBL phenotype. Of the 33 ESBL-producing E. coli isolates, 66.6% were resistant to tetracycline, 57.6% to sulfamethoxazole, 48.9% to nalidixic acid, 42.4% to ciprofloxacin and 33.3% to trimethoprim. Pulsed field gel electrophoresis (PFGE) results showed that the majority of E. coli isolates (16/33) and all Enterobacter spp. isolates (n = 5) were not clonally related (80% similarity cut value). The sequenced strains were observed to efficiently transfer their ceftazidime resistance to the recipient strain E. coli J53 at 37 °C (transfer rates: 101–102 transconjugants per donor cell). S1-PFGE showed that the transconjugants J53(p23MO01-T1) and J53(p23MO02-T1) had acquired plasmids of about 82 kb and 55 kb plasmids, respectively. According to WGS results, the E. coli isolate was assigned to ST162, while the C. freundii isolate was assigned to ST95. Conclusions: This study demonstrates that dairy animals are reservoirs of ESBL-producing bacteria.

Comparative Profiling of Milk Somatic Cells Proteomes Revealed Key Players in Mammary Immune Mechanisms During Mastitis in Tropical Sahiwal (Bos indicus) Cows.

Bovine mastitis poses a significant economic burden on the dairy industry worldwide. This pioneering proteomic study conducted a comparative profiling of milk somatic cell (SC) proteins contributing to mammary immune defense during subclinical and clinical mastitis (CM) in Sahiwal (Bos indicus) cows. Based on California mastitis test (CMT) scores, milk SC counts, differential leukocyte counts (DLCs), and bacteriological culture results, quarter milk SC samples were categorized into healthy (H), subclinical mastitis (SCM), and CM groups. Comparative proteome profiling of milk SCs was done using a label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic approach. The identified upregulated proteins in mastitis groups such as Vanin 2, Thioredoxin reductase-like selenoprotein T, Ceramidase, Lymphocyte antigen 75, Misshapen-like kinase 1 (MINK1), Thrombospondin 1, Macrophage scavenger receptor 1, Leupaxin, and Lipoamide acyltransferase, involved in immune responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed immune functions and pathways like antigen processing, complement cascades, extracellular matrix receptor interaction, efferocytosis, leukocyte migration, chemokine, peroxisome proliferator-activated receptors (PPARs), and transforming growth factor (TGF)-beta signaling. These findings provide essential information on proteomic profiling in milk SCs and contribute valuable insights into immune-related proteins regulated during mastitis in dairy cows. Further, validated proteins (Vanin 2, MINK1, and Thrombospondin 1) offer potential inflammatory biomarkers for early mastitis detection in dairy cows.

Prevalence, Risk Factors, and Antimicrobial Resistance of Staphylococcus and Streptococcus Species Isolated from Subclinical Bovine Mastitis.

Subclinical mastitis (SCM) is a prevalent serious disease among dairy cows worldwide. It poses a significant challenge to the dairy industry, animal welfare, and a threat to public health. The present study aimed to investigate the molecular detection, prevalence, and antimicrobial resistance of Staphylococcus spp. and Streptococcus spp. isolated from raw composite milk samples obtained from SCM dairy cattle in Bangladesh. A total of 612 quarters milk samples obtained from 153 cows were analyzed for SCM using the California Mastitis Test. Bacterial isolation and identification were carried out and bacterial species were confirmed using molecular polymerase chain reaction methods. Antimicrobial susceptibility testing was performed using disc diffusion method. The findings revealed that the prevalence of SCM was 70.3% (26/37), 35.95% (55/153), and 23.04% (141/612) in the herd, cow, and quarter levels, respectively. Among the positive samples, 92.7% (51/55) were Staphylococcus spp. (S. aureus, S. chromogenes, and S. simulans) and the remaining isolates were 7.3% (4/55) Streptococcus spp. (Streptococcus agalactiae and Streptococcus dysgalactiae). The most prevalent species was S. chromogenes, accounting for 67.3% (37/55). Antimicrobial susceptibility testing showed that 65.5% of isolates were susceptible to cefoxitin, whereas, 89.1% were resistant to penicillin. Overall, 12 isolates (21.8%) out of 55 were resistant to more than three classes of antimicrobials and were defined as multidrug-resistant isolates. Methicillin-resistance gene was detected in 61.1% of the cefoxitin-resistant isolates. A multivariate logistic regression analysis identified five potential risk factors including the lack of post-milking teat disinfection (OR: 3.06), absence of immediate feeding after milking (OR: 9.81), poor udder hygiene (OR: 7.83), tick infestation (OR: 13.76), and absence of dry cow therapy (OR: 3.31). The findings of the current study underscore the urgent requirement for targeted interventions, considering the identified factors to effectively manage and control SCM in dairy cows.

Prevalence of pathogens and antimicrobial resistance of isolated Staphylococcus spp. in bovine mastitis milk in South Korea, 2018-2022.

Staphylococcus spp. are one of the most predominant isolates in milk samples of dairy cows with mastitis worldwide. The aims of this study were to investigate the prevalence of bacterial pathogens in bovine mastitis milk samples in South Korea and the antimicrobial resistance profiles of staphylococcal isolates. In total, 1,245 strains were isolated from 1,260 mastitis quarter milk samples (with somatic cell counts ≥200,000 cells/mL) from 66 dairy farms between 2018 and 2022. The bacterial genus with the highest prevalence in bovine mastitis milk samples was Staphylococcus spp. (33.9%), followed by Streptococcus spp. (11.5%). S. aureus and non-aureus staphylococci (NAS) accounted for 11.0% and 89.0% of staphylococcal isolates, respectively. S. chromogenes was the most prevalent species among the 22 NAS species detected. S. aureus showed the highest resistance rates to penicillin (25.0%) and ampicillin (20.8%), whereas NAS showed the highest resistance rates to penicillin (18.3%), tetracycline (11.4%) and erythromycin (10.1%). Sixteen multidrug-resistant (MDR) isolates were only isolated from NAS, and the most commonly detected antimicrobial resistance gene in the 16 MDR isolates was mecA (75.0%), followed by tetK (62.5%), blaZ (50.0%), ermC (50.0%), and lnuA (43.8%). In conclusion, NAS were the most common isolates from mastitis milk in South Korea and MDR isolates carried a variety of antibiotic resistance genes. Our study suggests that continuous monitoring of the distribution and antimicrobial resistance in Staphylococcus spp., particularly NAS, is needed to improve the effectiveness of management and treatment strategies in dairy farms.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome

BackgroundDairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial.MethodsFour groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030.ResultsIn healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance.ConclusionsAt T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Comparing the Performance of Automatic Milking Systems through Dynamic Testing Also Helps to Identify Potential Risk Factors for Mastitis.

Automatic milking systems (AMSs) are revolutionizing the dairy industry by boosting herd efficiency, primarily through an increased milk yield per cow and reduced labor costs. The performance of milking machines, whether traditional or automated, can be evaluated using advanced vacuum meters through dynamic testing. This process involves scrutinizing the system and milking routine to identify critical points, utilizing the VaDia™ logger (BioControl AS, Rakkestad, Norway). Vacuum recordings were downloaded and analyzed using the VaDia Suite™ software under the guidance of a milking specialist. Access to data from AMSs across various manufacturers and herds facilitated a retrospective study aimed at describing and comparing key milk emission parameters for different AMS brands while identifying potential mastitis risk factors. Using the proper statistical procedures of SPSS 29.1 (IBM Corp., Armonk, NY, USA), researchers analyzed data from 4878 individual quarter milkings from cows in 48 dairy herds. Results indicated a significant variability in milking parameters associated with quarter milk yield and AMS brand. Notably, despite AMSs standardizing teat preparation and stimulation, this study revealed a surprisingly high frequency of two major mastitis risk factors-bimodality and irregular vacuum fluctuations-occurring more frequently than in conventional milking systems. This study, one of the few comparing different AMS brands and their performance, highlights the crucial role of dynamic testing in evaluating AMS performance under real-world conditions.

Increased pulsation ratio in rear quarters reduces machine-on time and milking on empty teats

Dairy cows usually store more milk in rear than in front quarters. At conventional machine milking this leads to an earlier cessation of milk flow in front than in rear quarters and hence unavoidable milking on empty front teats. We tested the hypothesis that an increased pulsation ratio in rear quarters reduces both milking on empty front teats and machine-on time. We have also tested if the different amplitude of cyclic vacuum fluctuations during milking with simultaneous vs. alternate pulsation within front and rear quarters, respectively, impacts milking characteristics. Ten Holstein dairy cows were milked twice daily at 14-h and 10-h milking intervals. Pulsation ratio in front quarters was 65:35, but was either set at 65:35, 70:30, 75:25 or 80:20 in rear quarters. Whole udder milk flow, claw vacuum, and mouthpiece chamber vacuum of all 4 quarters were recorded during milking. Teat tissue thickness of all 4 teats was measured with a cutimeter at 5 and 30 min after cluster detachment. Total milk yield did not differ among treatments, indicating complete udder emptying. The time of quarter milk flow (tQMF) of front quarters did not differ among treatments but decreased with increasing pulsation ratio in rear quarters. In addition, the faster milking of rear quarters at higher pulsation ratio caused higher peak and average milk flow rates, a shorter duration of milk flow decline, and a reduced machine-on time. The increased milk flow at a higher pulsation ratio did not cause a significant reduction of the claw vacuum at the used sample size. Teat tissue thickness did not significantly differ among treatments, likely because milking on empty teats did almost not occur in the rear quarters, and treatment settings did not differ in front quarters. Simultaneous pulsation caused higher amplitudes of cyclic vacuum fluctuation than alternate pulsation. However, milking characteristics did not differ between simultaneous and alternate pulsation. In conclusion, an increased pulsation ratio in rear quarters reduces machine-on time, increases milking performance, and reduces the time of milking on empty teats in front quarters, without an increased impact on teat tissue thickness.

Regulation of gene expression in milk secretory pathways during secretory activation in Holstein cows

Increased transcription of genes involved in lactose synthesis is a key feature of secretory activation of the mammary gland. We determined which genes are transcriptionally regulated during the transition from colostrum to milk in cows and whether more frequent milking of one udder half would increase milk yields and alter gene expression. We enrolled 12 Holstein cows immediately after calving, harvested colostrum, then randomly assigned one udder half to 12 h (2X) or 6 h (4X) milking intervals for 48 h after first milking. After 48 h, all quarters were milked twice daily until d7, when final quarter milk yields were collected. Yields and composition of colostrum and milk were recorded for each 12 h interval. After each milking, a strip sample of hind milk was collected for isolation of RNA from milk fat and quantification of selected transcripts via qPCR. Milk, milk fat, total protein, and lactose yields increased significantly over the initial 48 h and at d7 after calving. Quarters on 4X treatment produced more milk than the contralateral 2X quarters. Genes upregulated concomitantly with milk yields encoded α-lactalbumin (LALBA), β-1,4-galactosyltransferase (B4GALT1), fatty acid synthase (FASN), β casein (CSN2), and folate receptor α (FOLR1). Downregulated genes encoded monosaccharide transporters (SLC2A3, SLC2A8, SLC35A2) and enzymes involved in galactose synthesis (HK1, PGM1, GALE). Three genes were initially downregulated but later upregulated at d7 (LPIN1, SLC2A1, UGP2). Notably, milking frequency had no effect on gene expression. Sequential upregulation first of genes encoding the former enzymes, then of UGP2, may be necessary for copious milk production. However, the local effects of milking frequency on milk production during lactogenesis do not appear to be controlled by transcription of these genes related to lactose synthesis.

Distribution of Bovine Mastitis Pathogens in Quarter Milk Samples from Bavaria, Southern Germany, between 2014 and 2023-A Retrospective Study.

The objective of this study was to investigate the distribution of mastitis pathogens in quarter milk samples (QMSs) submitted to the laboratory of the Bavarian Animal Health Service (TGD) between 2014 and 2023 in general, in relation to the clinical status of the quarters, and to analyze seasonal differences in the detection risk. Each QMS sent to the TGD during this period was analyzed and tested using the California Mastitis Test (CMT). Depending on the result, QMSs were classified as CMT-negative, subclinical, or clinical if the milk character showed abnormalities. Mastitis pathogens were detected in 19% of the QMSs. Non-aureus staphylococci (NAS) were the most common species isolated from the culture positive samples (30%), followed by Staphylococcus (S.) aureus (19%), Streptococcus (Sc.) uberis (19%), and Sc. dysgalactiae (9%). In culture-positive QMSs from CMT-negative and subclinically affected quarters, the most frequently isolated pathogens were NAS (44% and 27%, respectively), followed by S. aureus (25% and 17%, respectively) and Sc. uberis (8% and 22%, respectively). In QMSs from clinically affected quarters, the most frequently isolated pathogens were Sc. uberis (32%), S. aureus (13%), Sc. dysgalactiae (11%), and Escherichia (E.) coli (11%). The distribution of NAS and Sc. uberis increased throughout the study period, while that of S. aureus decreased. From June to October, QMSs from subclinically affected quarters increased and environmental pathogens, such as Sc. uberis, were detected more frequently. In conclusion, this study highlights the dynamic nature of the distribution of mastitis pathogens, influenced by mastitis status and seasonal factors. Environmental pathogens still play an important role, especially in clinical mastitis and seasonal dependency, with the number of positive samples continuing to increase. It is therefore essential to continue mastitis control measures and to regularly monitor the spread of mastitis pathogens in order to track trends and adapt targeted prevention measures.

Effects of rumen-protected niacin on inflammatory response to repeated intramammary lipopolysaccharide challenges

Nutritional strategies that improve an animal's resilience to various challenges may improve animal health and welfare. One such nutrient is niacin which has reduced inflammation in mice, humans, and swine; however, niacin's anti-inflammatory effects have not been investigated in cattle. Our objective was to determine whether rumen-protected niacin (RPN) alters lactating dairy cows' inflammatory response to intramammary lipopolysaccharide (LPS) challenges, whether RPN resulted in any carry-over effects, and whether repeated LPS challenges result in signs of immune tolerance or innate immune training. Twenty healthy, late-lactation Holstein cows (232 ± 65 d in milk; 39 ± 5.8 kg/d of milk) were enrolled in a randomized complete block experiment which lasted 70 d. Cows received 26 g/d of RPN or no top-dress (CON) for the first 42 d of the experiment. During the final milking of d 27 and 55, cows were challenged in their rear-right mammary gland (RR) with 100 µg of LPS suspended in 5 mL of phosphate buffered saline. Milk yield, milk conductivity, and feed intake were measured daily. Milk composition was measured on d 14, 23, 24, 30, 37, 45, and 52. Blood samples were collected at 0, 8, 12, 24, 48, 72, 96, and 120 h after each LPS challenge, whereas RR quarter milk samples were collected at 0, 8, 16, 24, 48, 72, 96, 120, 144, and 168 h after each LPS challenge. Body temperature was measured continuously during each challenge with an intravaginal thermometer. Linear mixed models with repeated measures were used to analyze the results. Before LPS challenge, RPN did not affect feed intake or milk production, but it reduced SCS (1.24 ± 0.41 vs. 0.05 ± 0.45). After challenge, RPN did not affect feed intake, milk production, milk composition, SCS, body temperature, plasma glucose, or plasma insulin concentrations. Our results suggest RPN reduced peak plasma haptoglobin and lipopolysaccharide binding protein (LBP) during the 1st LPS challenge. Plasma haptoglobin tended to be less after the 2nd challenge for cows previously supplemented RPN while LBP was similar for each treatment group after the 2nd challenge. The 2nd LPS challenge resulted in decreased plasma haptoglobin compared with the 1st LPS challenge, suggestive of tolerance but it also induced a greater peak SCS than the 1st LPS challenge. Our results suggest that repeated LPS challenges promote a systemic tolerance but heightened local response to LPS-induced mastitis. Feeding RPN reduced SCS before challenge and reduced plasma acute phase proteins after challenge suggesting that RPN may reduce systemic inflammation without altering the local inflammatory responses.

Presence of pathogen DNA in milk harvested from quarters is associated to changes in cows’ milk yield and composition

BackgroundIntramammary infection is the result of invasion and multiplication of microorganisms in the mammary gland and commonly leads to mastitis in dairy animals. Although much has been done to improve cows’ udder health, mastitis remains a significant and costly health issue for dairy farmers, especially if subclinical. In this study, quarter milk samples from clinically healthy cows were harvested to detect pathogens via quantitative PCR (qPCR) and evaluate changes in individual milk traits according to the number of quarters infected and the type of microorganism(s). A commercial qPCR kit was used for detection of Mycoplasma bovis, Mycoplasma spp., Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Prototheca spp., Escherichia coli, Klebsiella spp., Enterococcus spp. and Lactococcus lactis ssp. lactis. Quarter and pooled milk information of 383 Holstein, 132 Simmental, 129 Rendena, and 112 Jersey cows in 9 Italian single-breed herds was available.ResultsAmong the cows with pathogen(s) present in at least 1 quarter, CNS was the most commonly detected DNA, followed by Streptococcus uberis, Mycoplasma bovis, and Streptococcus agalactiae. Cows negative to qPCR were 206 and had the lowest milk somatic cell count. Viceversa, cows with DNA isolated in ≥ 3 quarters were those with the highest somatic cell count. Moreover, when major pathogens were isolated in ≥ 3 quarters, milk had the lowest casein index and lactose content. In animals with pathogen(s) DNA isolated, the extent with whom milk yield and major solids were impaired did not significantly differ between major and minor pathogens.ConclusionsThe effect of the number of affected quarters on the pool milk quality traits was investigated in clinically healthy cows using a commercial kit. Results remark the important negative effect of subclinical udder inflammations on milk yield and quality, but more efforts should be made to investigate the presence of untargeted microorganisms, as they may be potentially dangerous for cows. For a smarter use of antimicrobials, analysis of milk via qPCR is advisable – especially in cows at dry off - to identify quarters at high risk of inflammation and thus apply a targeted/tailored treatment.

Antimicrobial resistance of Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus canis in quarter milk samples from Bavaria, Southern Germany, between 2012 and 2022

The objective of this study was to analyze the in vitro antimicrobial resistance (AMR) of Streptococcus (Sc.) dysgalactiae, Sc. agalactiae, and Sc. canis over a 10-year period from 2012 to 2022 against the most commonly used antimicrobial agents. For this purpose, all quarter milk samples (QMS) submitted to the milk laboratory of the Bavarian Animal Health Service (TGD) were analyzed. Each QMS was tested using the California Mastitis Test (CMT) and categorized as negative (N), subclinical (S), or clinical (C) mastitis if the milk character was abnormal. Samples with Sc. dysgalactiae, Sc. agalactiae, or Sc. canis were included and a subset of isolates were further tested for in vitro antimicrobial resistance by breakpoint analysis with broth microdilution. Sc. dysgalactiae (61%, n = 65,750) was the most abundant pathogen among those 3 species, followed by Sc. agalactiae (28%, n = 30,486), and Sc. canis (11%, n = 11,336). All 3 species showed the highest resistance to the same 4 antimicrobial agents: erythromycin, marbofloxacin, pirlimycin, and cefalexin/kanamycin with varying degrees of resistance. Throughout the study period, Sc. dysgalactiae, Sc. agalactiae, and Sc. canis were largely susceptible to the remaining antimicrobial agents tested (penicillin, amoxicillin-clavulanate, oxacillin, cefazolin, cefoperazone, cefquinome). Only less than 14% of isolates of Sc. dysgalactiae and Sc. canis were resistant against any of the antimicrobials tested. Sc. agalactiae was the species with the highest percentage of resistant isolates. While the percentage of resistant isolates from Sc. canis and Sc. dysgalactiae decreased, the percentage of resistant Sc. agalactiae isolates increased since 2017. In summary, most isolates were not resistant to the most commonly used antimicrobial agents for mastitis therapy, including β-lactam antibiotics and penicillin should remain the first-choice therapy against streptococcal mastitis.

In vitro antimicrobial resistance of Escherichia coli, Serratia marcescens, Klebsiella oxytoca, and Klebsiella pneumoniae on Bavarian dairy farms between 2014 and 2022

The objective of this study was to describe the prevalence of antimicrobial resistance of E. coli, K. oxytoca, K. pneumoniae, and S. marcescens from quarter milk samples submitted to the udder health laboratory of the Bavarian Animal Health Services (TGD) in Southern Germany between 2014 and 2022. All samples were tested with the California Mastitis Test and analyzed with a standard microbroth dilution to determine the minimum inhibitory concentrations (MIC). The antimicrobials tested were amoxicillin/clavulanate, cefazoline, kanamycin/cefalexin, cefoperazone, cefquinome, and marbofloxacin. Breakpoints were chosen in accordance with CLSI. Over the study period, E. coli, K. oxytoca, and K. pneumoniae showed only few resistances to all antimicrobials tested. For those pathogens MIC 50 and MIC 90 were below breakpoint for all antimicrobials except cefoperazone over the 9 years. A decrease in MIC could be seen for E. coli and K. oxytoca for all of the antimicrobials. While the MIC for K. pneumoniae stayed more stagnant, the prevalence of resistance still decreased overall. S. marcescens isolates were proven intrinsically resistant to amoxicillin/clavulanate and cefazolin and while in vitro resistances were low for all other antimicrobials tested, S. marcescens tended toward higher MIC for most of the antimicrobials over the years. Over time, there was also an overall increase in the number of isolates for all 4 pathogens per year. Starting 2018 there was steep increase in the number of isolates particularly from clinical cases. This jump in numbers coincided with a change of the regulation for veterinary drug prescriptions in Germany in 2018 that required, among other things, antimicrobial resistance testing before a change of antibiotics in the course of treatment and the use of critically important antimicrobials. Overall, while the pathogens increased in numbers, the prevalence of their antimicrobial resistance remained low.

Effect of milk stasis on mammary gland involution and the microRNA profile

The presence of an autocrine factor in milk that can trigger mammary gland involution was proposed more than 50 yr ago. To provide evidence for the existence of one or more autocrine factors, 10 multiparous cows in late lactation were quarter-milked for 7 d. Following this baseline period, the right front quarter of each cow was left unmilked, and the other quarters were milked for 7 d. Before the last milking of that period, milk (mammary secretions) was collected aseptically from both front quarters. After that milking, 250 mL of the collected samples were infused in the cows' respective rear quarters. No quarters were milked for the following 7 d (milk stasis period), and quarter milking was then resumed in all quarters for the last 7 d of the experiment (remilking period). Quarter milk samples were collected during the baseline period, before the milk stasis period, and during the remilking period. These samples were used for measuring milk components and the concentration of involution markers (SCC, BSA, and lactoferrin). Samples of mammary secretions were collected manually from the quarters during the milk stasis period for involution marker determination. We extracted RNA from samples collected from front quarters before the last milking before the milk stasis period for microRNA (miRNA) determination. As anticipated, the longer milk stasis period implemented for the right front quarter resulted in a more advanced involution than in the left front quarter, based on the concentration of involution markers in the mammary secretions, lower milk production recovery, and changes in milk composition during the remilking period. All 3 involution marker concentrations in the mammary secretions increased in both rear quarters, but were greater in the right quarter secretions than in the left quarter secretions. Resuming milking reinitiated milk production in all quarters, but milk production recovery in the right rear quarters was less robust than that in the left rear quarters (54.3 ± 1.4% vs. 61.6 ± 1.4%, respectively). Milk from the quarters infused with mammary secretions (right rear) had a lower lactose content, but a higher milk protein content and higher SCC than the quarters infused with milk. We detected a total of 359 miRNAs, 76 of which were differentially expressed in milk and mammary secretions. Expression of bta-miR-221 and bta-miR-223 was upregulated in mammary secretions 34- and 40-fold, respectively. The results of the present experiment support the contention that milk stasis leads to the accumulation of one or more factors that trigger involution. The results also indicate that milk stasis leads to changes in the miRNA profile of the milk, but whether such changes are a cause or a consequence of the involution process remains to be established.

The Influence of Milk Leakage, Udder Pressure and Further Risk Factors on the Development of New Intramammary Infections during the Dry Period of Dairy Cows.

Prevention of new intramammary infection (NIMI) during the dry period (DP) is essential to prevent the development of mastitis in dairy cows. To investigate risk factors for NIMI, 212 cows, comprising a total of 848 udder quarters, were examined in this study. Quarter milk samples were taken on the day of drying off and 7 ± 3 days after calving. Cow- and quarter-level associated risk factors were assessed at the beginning of the DP and after calving. In total, 7.1% of the udder quarters developed an NIMI between the samplings. Non-aureus staphylococci (40.4%) and Gram-negative pathogens (22.8%) were most frequently the cause of NIMI. The observed milk leakage prevalence was 16.7%, with a peak 24 h after drying off. Simultaneously, the udder pressure peaked 24 h after drying off. A significant correlation between milk yield on the day before drying off and milk leakage could be proven. Cows with quarters leaking milk produced an average milk yield of 28.32 kg on the day before drying off. Generalised linear mixed models and odds ratios were calculated to determine the significant risk factors for NIMI during the DP and early lactation. Quarters leaking milk had 3.4 higher odds for NIMI between the samplings compared to quarters without milk leakage. Quarters from cows with dirty udders had 3.1 higher odds of developing an NIMI between the samplings compared to quarters from cows with clean udders. The results of this study demonstrate the importance of dry cow management before drying off and during the critical period of active involution of the udder tissue.

Milk yield and quality at udder quarter level are influenced by quarter position, pathogen, and somatic cell score

There is a paucity of information about milk composition and quality at the quarter level from complete milking of the udder, mainly because of the difficulties of sampling procedures which imply the adoption of a vacuum system connected to four independent buckets. The objective of this study was to evaluate the effect of quarter position, intramammary infection status, and somatic cells on milk yield and composition at the mammary quarter level of Simmental cows. Milk samples were collected during three consecutive days, after milking individual mammary quarters of 10 lactating cows separately, for a total of 120 quarter milk samples. Samples were analysed for composition, somatic cell count (SCC), differential SCC, and bacterial culture. Somatic cell count was log-transformed to somatic cell score (SCS) to achieve normality of distribution. Sources of variation of milk yield and quality traits were investigated using a linear mixed model which included the fixed effects of quarter position (1 to 4), pathogen presence (positive or negative), and SCS class (low, medium-low, medium-high, and high). The interaction between cow and quarter position was included as random effect. Quarter position significantly affected milk yield, with rear udder quarters yielding more milk compared to their front counterparts, but with negligible effects on milk composition. Classes of SCS influenced milk composition, in a way that quarters with high SCS had lower lactose content and higher differential SCC. This study is a contribution towards the possibility to better understand physiological processes at cow udder quarter level.

A Temporal Study on Incidence of Bovine Mastitis in Haryana, India

Mastitis affects dairy animal’s productivity and causes financial losses for dairy farmers in India and across the world. In this study, a total of 52,494 quarter milk samples from 14,381 bovines were screened for the primary microorganisms causing mastitis in Hisar and adjoining districts of Haryana and their antibiotic sensitivity patterns were analyzed. The cultural positivity from subclinical form of mastitis was observed as 86.32% and 87.73% from cows and buffaloes, respectively while that from clinical mastitis was 87.36% and 87.57%. The major Gram positive bacterial pathogens associated with mastitis in the entire study period were found as Staphylococcus species with an average incidence rate of 45.53% and 44.1% from cows and buffaloes, respectively. The Streptococcus species were found to be 33.76% and 29.94% of total isolates. Escherichia coli were the most predominant Gram negative bacteria isolated (17.37% and 13.85%), thereafter Klebsiella spp. (5.54% and 5.19%) from both cows and buffaloes. A significant proportion of clinical cases of mastitis were chronic in nature from both the species of bovines. The incidence of mastitis with respect to different lactation number and lactation months was found as significant. The highest incidences of mastitis was observed in the first lactation among buffaloes (20.69%), while the maximum incidences were observed in second and third lactation in cows with 16.59% and 16.99%, respectively. However, in both the species maximum occurrence of mastitis was observed during the first lactational month. The antibiotic sensitivity patterns of most of the isolates had shown higher sensitivity towards enrofloxacin and gentamicin, while the penicillin had shown least sensitivity. The knowledge regarding the mastitis causing pathogens and their sensitivity pattern in Hisar and adjoining districts of Haryana enables the veterinarians to adopt for the proper treatment protocols and dairy farmers to assure optimal health, welfare and productivity of bovines in the State, in turn reducing antimicrobial resistance.

Pathogen group-specific risk factors for intramammary infection in water buffalo.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Antimicrobial resistance profiling of coagulase negative staphylococci isolated from bovine mastitis

Mastitis plays a crucial role in the economics of dairy industry by deteriorating the quality and quantity of milk produced, as well as endangering the animal health and welfare. The objective of this study was to investigate the antimicrobial resistance (AMR) profile of coagulase negative staphylococci (CNS) isolated from bovine mastitis. Microbiological evaluation by morphological, cultural and biochemical characterisation as well as by monoplex polymerase chain reaction (PCR) of the 83 quarter milk samples revealed that CNS were the most predominant bacteria (32.53%). It was also concluded that most of the CNS were resistant to multiple antibiotics. The phenotypic and genotypic AMR profiling was done using in vitro disc diffusion assay and PCR, respectively, to identify the resistance pattern towards penicillin, methicillin, tetracycline and enrofloxacin. The results depicted a significant difference between the phenotypic and genotypic resistance of CNS against penicillin, methicillin and tetracycline. This outcome on the interaction of phenotypic and genotypic AMR profiling is intriguing and opens a huge scope for future studies on the transcriptomic and proteomic aspects of drug resistance. A better knowledge of the AMR profile guides the dairy producers in developing suitable timely intervention strategies for the economic management of mastitis, which in turn helps in tackling AMR and reduces the threat of its zoonotic transmission.

Pathogen-specific patterns of milking traits in automatic milking systems

Early detection of intramammary infection (IMI) can improve animal health and welfare in dairy herds. The implementation of sensors and automatic milking systems (AMS) in dairy production inherently increases the amount of available data and hence also the potential for new approaches to mastitis management. To utilize the full potential of data from AMS and auxiliary sensors, a better understanding of physiological and pathological changes in milking traits associated with different udder pathogens may be imperative. This observational study aimed to investigate pathogen-specific patterns in milking traits recorded in AMS. The milking traits included; online somatic cell count (OCC), electrical conductivity (EC), milk yield (MY), and average milk flow rate (AMF). Data were collected for a study period of 2 years and included 101 492 milkings from 237 lactations in 169 cows from one farm. Measurements of OCC were recorded at cow-level and data on EC, MY, and AMF were obtained at quarter-level. In addition to the data obtained from the AMS, altogether 5756 quarter milk samples (QMS) were collected. Milk samples were obtained monthly for bacteriological culturing. We included findings of 13 known mastitis pathogens to study pathogen-specific patterns in milking traits. These patterns were compared with those in a baseline group consisting of cows that did not have any positive milk culture results throughout the lactation period. Patterns of the milking traits are described for all positive samples both across 305 d in milk (DIM), and in the 15-d period before a positive bacteriological sample. The association between a positive sample and the milking traits (ln(OCC), EC-IQR; the ratio between the quarter with the highest and the quarter with the lowest level of EC, and MY) for the 15 d before the detection of a pathogen was assessed using mixed effects linear regression models. All pathogens were associated with alterations in the level and variability of ln(OCC) relative to lactations with no positive bacteriological samples. A positive sample for Staph. aureus was associated with increased values for MY during the 15 d before a positive diagnosis. It is biologically plausible to interpret changes in OCC and EC-IQR as consequences of an intramammary infection (IMI), while higher MY in bacteriologically-positive cows is most likely linked to the increased risk of infection in high-yielding cows. In this study, the most notable changes in the traits (OCC and EC-IQR) were observed for Staph. aureus and Strep. dysgalactiae, followed by Strep. simulans, Strep. uberis, and Lactococcus lactis. Even if we did not detect significant associations between positive bacteriology and EC-IQR, visual assessment and descriptive statistics indicated that there might be differences suggesting that it could be an informative trait for detecting infection when combined with OCC and possibly other relevant traits using machine learning algorithms.